New Bicistronic TALENs Greatly Improve Genome Editing.

Genome editing has become one of the most powerful tools in present-day stem cell and regenerative medicine research, but despite its rapid acceptance and widespread use, some elements of the technology still need improvement. In this unit, we present data regarding the use of a new, more efficient type of transcription activator-like effector nuclease (TALEN) for gene editing. Our group has generated bicistronic genes in which classical TALEN coding sequences are linked by 2A elements to different reporter molecules, such as fluorochromes (TALEN-F) or membrane receptors (TALEN-M). This structure results in two proteins transcribed from the same transcript, of which the second (the reporter) can be used as the target for selection by fluorescence-assisted cell sorting (FACS) or magnetic-activated cell sorting (MACS). The application of these new TALEN genes allows a rapid enrichment of cells in which both members of the TALEN pair are active, thus eliminating the need for lengthy selection in culture and laborious characterization of a large number of clones. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of new TALENs Basic Protocol 2: Genome editing using TALEN-F Alternate Protocol 1: Generation of TALEN-M Support Protocol 1: mRNA in vitro transcription (IVT) of TALEN-T2A-reporter expression vector Alternate Protocol 2: Editing of primary T cells using TALEN-M Basic Protocol 3: Verifying gene editing Support Protocol 2: Rapid expansion protocol for edited T-cells.

Safety and effectiveness of sodium colistimethate-loaded nanostructured lipid carriers (SCM-NLC) against P. aeruginosa: in vitro and in vivo studies following pulmonary and intramuscular administration.

The usefulness of nanotechnology to increase the bioavailability of drugs and decrease their toxicity may be a tool to deal with multiresistant P. aeruginosa (Mr-Pa) respiratory infections. We describe the preparation and the in vivo efficacy and safety of sodium colistimethate-loaded nanostructured lipid carriers (SCM-NLC) by the pulmonary and intramuscular routes. Nanoparticles showed 1-2 mg/L minimum inhibitory concentration against eight extensively drug-resistant P. aeruginosa strains. In vivo, SCM-NLC displayed significantly lower CFU/g lung than the saline and similar to that of the free SCM, even the dose in SCM-NLC group was lower than free SCM. There was no tissue damage related to the treatments. Biodistribution assessments showed a mild systemic absorption after nebulization and a notorious absorption after IM route. Altogether, it could be concluded that SCM-NLC were effective against P. aeruginosa in vivo, not toxic and distribute efficiently to the lung and liver after pulmonary or intramuscular administrations.

Generation of two induced pluripotent stem cells lines from Mucopolysaccharydosis IIIA patient: IMEDEAi004-A and IMEDEAi004-B.

Mucoplysaccharydosis IIIA (MPSIIIA) is the most severe form of Sanfilippo syndrome. Skin fibroblasts from a MPSIIIA compound heterozygous (E447K/R245H) patient were nucleofected with four OriP/EBNA1-based episomal plasmids containing: OCT3/4, SOX2, KLF4, L-Myc, LIN28, BCL-xL and shp53. The two iPSCs lines generated carry both sulfamidase enzyme (SGSH) mutations, are free of plasmid integration, have normal karyotype, express pluripotency-associated markers and are able to differentiate into the three germ layers.

Generation of two induced pluripotent stem cell (iPSC) lines from p.F508del Cystic Fibrosis patients.

Cystic Fibrosis (CF) is a monogenic, lethal disease caused by mutations in the cystic fibrosis transmembrane conductance (CFTR) gene. Here we report the production of CF-iPS cell lines from two different p.F508del homozygous female patients (Table 1). Two different primary cell types, skin fibroblasts and keratinocytes, were transfected with retroviral cocktails containing four: c-MYC, KLF4, OCT4 and SOX2 (MKOS) or three: KLF4, OCT4 and SOX2 (KOS) reprogramming factors. Two fibroblast-derived MKOS lines are described in the main text. The lines carry the p.F508del mutation, have a normal karyotype, express pluripotency markers and are able to differentiate into the three germ layers.

Pulmonary delivery of tobramycin-loaded nanostructured lipid carriers for Pseudomonas aeruginosa infections associated with cystic fibrosis.

Among the pathogens that affect cystic fibrosis (CF) patients, Pseudomonas aeruginosa is the most prevalent. As a way to fight against this infection, nanotechnology has emerged over the last decades as a promising alternative to overcome resistance to antibiotics in infectious diseases. The goal of this work was to elaborate and characterize lipid nanoparticles for pulmonary delivery of tobramycin. Tobramycin-loaded nanostructured lipid carriers (Tb-NLCs) were prepared by hot melt homogenization technique. In addition, nanoparticles labeled with infrared dye (IR-NLCs) were used to investigate their in vivo performance after pulmonary administration. Tb-NLCs displayed a mean diameter size around 250 nm, high drug encapsulation (93%) and sustained release profile. Tb-NLCs showed to be active against clinically isolated P. aeruginosa. Moreover, Tb-NLCs did not decrease cell viability and were able to overcome an artificial mucus barrier in the presence of mucolytics agents. During the in vivo assay, IR-NLCs were administered to several mice by the intratracheal route using a Penn Century device. Next, the biodistribution of the nanoparticles was analyzed at different time points showing a wide nanosystem distribution in the lungs. Altogether, tobramycin-loaded NLCs seem to us an encouraging alternative to the currently available CF therapies.